Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids

Summary Human induced pluripotent stem cell (hiPSC)-derived hair-bearing skin organoids offer exciting new possibilities for modeling diseases like epidermolysis bullosa (EB). These inherited diseases affect 1 in 30,000 people worldwide and result from perturbed expression and/or structure of components of the epidermal-dermal junction (EDJ). To establish whether hiPSC-derived skin organoids might be able to capture salient features of EB, it is thus important to characterize their EDJ. Here, we report successful generation of hair-bearing skin organoids from two hiPSC lines that exhibited fully stratified interfollicular epidermis. Using immunofluorescence and electron microscopy, we showed that basal keratinocytes in organoids adhere to laminin-332 and type IV collagen-rich basement membrane via type I hemidesmosomes and integrin β1-based adhesion complexes. Importantly, we demonstrated that EDJs in organoids are almost devoid of type VII collagen, a fibril that mediates anchorage of the epidermis to dermis. This should be considered when using skin organoids for EB modeling.


INTRODUCTION
Skin integrity depends on the epidermal-dermal junction (EDJ), the interface between basal keratinocytes of the epidermis and stroma of the dermis. Basal keratinocytes adhere to the underlying basement membrane (BM), an extracellular matrix (ECM) sheet, composed primarily of laminin and type IV collagen (ColIV) networks (Burgeson and Christiano, 1997). This adhesion is mediated by integrins, transmembrane ab heterodimer proteins that cluster to adhesion structures on the basal plasma membrane of keratinocytes. In physiological conditions, keratinocytes highly express collagen-binding integrin a2b1, laminin-332/-511-binding a3b1, and laminin-332-binding a6b4. The adhesive integrity of the EDJ largely depends on a6b4-containing adhesion structures called hemidesmosomes (HDs). In contrast to simple epithelia (e.g., intestine), which express type II HDs consisting solely of the integrin a6b4 and plectin, keratinocytes in the EDJ adhere via type I HDs that additionally contain tetraspanin CD151 and bullous pemphigoid antigens 180 (BP180) and 230 (BP230) (Margadant et al., 2010). Additionally, BM is connected to the dermal connective tissue via anchoring fibrils, consisting of ColVII (Burgeson and Christiano, 1997).
Aside from its function in epidermal adhesion, the EDJ also regulates the permeability barrier of the skin and plays a role in epithelial-mesenchymal interactions and signal transduction. Its pivotal role in skin physiology is reflected in severe pathologies that occur when the function of EDJ components is impaired. This is the case in, for example, epidermolysis bullosa (EB), which is caused by, among others, mutations in genes encoding components of HDs and associated filaments. These include mutations in the COL7A1 gene (coding for ColVII) causing dystrophic EB, a severe blistering disease (Turcan and Jonkman, 2015). Furthermore, EDJs, particularly BMs and integrins, play an important role in initiation and progression of skin cancers (Chang and Chaudhuri, 2019;Ramovs et al., 2016).
The generation of hair-bearing skin organoids derived from human pluripotent stem cells (hPSCs) has recently been described . These clearly offer exciting new opportunities for skin disease modeling, regenerative medicine, and developmental research. However, to grasp the full potential of this model, it is essential to understand the structure and developmental stage of EDJs formed in the skin organoids. This is especially important since their gene expression profile and hair morphology indicate that they represent mid-gestational skin, a fetal stage during which the main components of the EDJ gradually begin to develop (Nicholas and Jacques, 2005). In addition, inherent differences between hPSC lines can lead to variable outcomes (Lee and Koehler, 2021) and therefore require the validation of protocols with independent cell lines.
In this study, we report the generation of hair-bearing skin organoids from two male human induced pluripotent stem cell (hiPSC) lines and address the organization of their EDJ using immunofluorescence (IF) and transmission electron microscopy (TEM).

RESULTS AND DISCUSSION
Successful generation of hair-bearing skin organoids from two hiPSC lines Using two independent control hiPSC lines (LUMCi045-A1 and LUMCi046-A1), we were able to recapitulate the in vitro skin organogenesis described by Lee et al. (2020) (Figure 1A). Consistent with the original protocol, the majority of skin organoids became bipolar at around day 30 of directed differentiation, with an opaque cell mass (''tail'') at the pole opposite to the translucent epidermal cyst (''head'') ( Figure 1A). At around day 50, the epithelium was composed of a basal layer of keratin 5 (KRT5) + keratin 15 (KRT15) + cells, an intermediate KRT5 low layer, and a KRT5 low KRT15 high periderm-like layer ( Figure 1B). Hair placodes were observed at around day 60, and fully grown hair follicles (HFs) developed after 100 days in culture ( Figures 1A-1C). At day 130, 78.9% and 75.7% of the skin organoids derived from LUMCi045-A1 and from LUMCi046-A1 displayed HFs, with a mean number of 18 and 16 HFs per organoid, respectively ( Figure 1D). HFs showed similar organization to that previously reported in organoid-derived HFs, occasionally containing sebumproducing sebaceous glands (Figures 1C, S1A, and S1B) and innervation as illustrated by TUJ1 + neuronal processes and TUJ1 + Merkel-like cells ( Figure S1C) . When pigmented, the matrix region of the HFs and interfollicular epidermis were enriched with PMEL + melanocytes (Figures S1D and S1E). Skin organoids were rich in vimentin + dermal fibroblasts, which, much like mid-gestational fetal skin, clustered to FAP high papillary fibroblasts in the upper dermis and CD90 high FAP low reticular fibroblasts in the lower dermis ( Figures 1E and S1D) (Korosec et al., 2019). Consistent with the previous report, we also observed hyaline cartilage in all organoids analyzed (Figure S1F) .
hiPSC-derived skin organoids form a stratified interfollicular epidermis At day 130, skin organoids formed a stratified epidermis, which resembles the stratification of the adult human skin: basal keratinocytes express KRT5 and KRT14, and suprabasal keratinocyte layers contain KRT1 and KRT10, with a terminally differentiated loricrin-positive cornified layer marking the upper end of the epidermis ( Figures 1C  and 1F). Such complete stratification of organoid interfol-licular epidermis was shown previously when mature organoids were grafted in a mouse model , but our data support the notion that organoid grafting may not be necessary for short-term experiments that require mature stratification. Nonetheless, it is important to note that organoid grafting in mice leads to the formation of rete-ridge-like structures, which are present in human skin but which neither we nor  observed in the epidermis of skin organoids ( Figure 1F). As rete ridges develop after week 19 of gestation ( Figure 1F), this likely indicates increased maturity of the model after grafting. Furthermore, mechanical stress has been shown to play an important role in the development of rete ridges (Penrose and Ohara, 1973;Xiong et al., 2013), thus their absence might also reflect the lack of mechanical stress in the in vitro culture.
BM underlies the epidermis of hiPSC-derived skin organoids Basal keratinocytes adhere to the BM, an ECM sheet composed of ColIV and laminin networks, which can be detected as early as week 7 of fetal development (Hertle et al., 1991). Epidermal BM predominantly contains laminins-332 and -511, of which laminin-332 functions as a main anchoring filament for basal keratinocytes (Burgeson and Christiano, 1997;Pozzi et al., 2017). In organoids, both laminin-332 and ColIV showed homogeneous expression, restricted to the BM ( Figures 1F and 2A). Consistent with their role in adhesion to the BM, all subunits of the major epidermal integrins could be detected at the EDJ in the epidermal cyst of organoids: a3, a6, b4, and b1 ( Figures 2B and 2C). In adult skin, integrin a6b4 mainly localizes to the basal membrane of basal keratinocytes, whereas its expression is pancellular early during fetal development, with progressive restriction to the basal membrane from week 10 of gestation (Hertle et al., 1991). The stratified epidermis of immature organoids (day 55) resembles early stages of skin development with a strong expression of integrin a6b4 also at the lateral and apical membranes of basal keratinocytes ( Figure S2); this becomes more restricted to the basal membrane in mature organoids (day 130), reflecting the organization of human skin from mid-gestation onward ( Figure 2B). Integrin a3b1 can be observed at basal and lateral membrane of keratinocytes at EDJ of organoids, which is in line with its localization in mature epidermis ( Figure 2B, white arrows) (Zuidema et al., 2020).
Next, we examined the expression of ColVII, which anchors BM to the dermis, using polyclonal and monoclonal antibodies. While homogeneous and abundant in adult skin, ColVII expression was rather heterogeneous and barely detectable along the EDJ of skin organoids. By contrast, all other ECM components of the EDJ that were  (B) Representative confocal images of organoid and human skin sections stained for laminin-332 in combination with keratin 5 (KRT5) and integrin b4 subunit (left panels), integrin b1 and integrin b4 subunits (middle panels), or integrin a6 and integrin a3 subunits (right panels). The distribution of laminin-332 and integrin subunits a3, a6, b4, and b1 in skin organoids is similar to that in adult and midgestational fetal human skin. Arrows point to the localization of integrin a3b1 to cell-cell contacts. Scale bars: 40 mm. (C) Confocal images of organoid whole mounts stained with anti-KRT5 and anti-b4 integrin antibodies (left panels) or with anti-vimentin and anti-a3 and -a6 integrin subunit antibodies (right panels). Note the homogeneous distribution of integrins a3b1 and a6b4 to the polarized head of organoids. Scale bars: 200 mm. Organoids are imaged at day 130. Dashed boxes are magnified. Confocal images are presented as maximum projected z stacks. examined were clearly detectable, albeit at reduced levels compared with human skin (Figures 3A, 3B, and S3A). In line with this, the expression of COL7A1 was lower in organoids compared with adult human skin when normalized to the expression of two chains encoding laminin-332 and two chains encoding ColIV ( Figure S3B).
The low levels of ColVII are remarkable considering that weak expression of this protein has been detected as early as at 8 weeks of gestation in fetal skin and that homogeneous interfollicular expression has been observed from 13 weeks of gestation onward (Karelina et al., 2000;Ryynänen et al., 1992). Indeed, analysis of 19-week-old fetal skin revealed comparable levels of ColVII to those in adult human skin ( Figures 3A and 3B). Given that skin organoids resemble fetal skin of gestational week 18 , low and heterogeneous levels of expression of ColVII were not expected. To rule out any specific defects in the hiPSC lines used in this study, we quantified the expression of ECM components in organoids generated from previously published female hiPSC line WT2. The levels of ColVII in WT2 organoids were similar to those observed in LUMCi045-A1 and LUMCi046-A1 ( Figures S3C and S3D). We further differentiated LUMCi045-A1 and LUMCi046-A1 directly to keratinocytes and compared their levels of ColVII with those in primary human keratinocytes. When grown on glass coverslips, hiPSC-derived and primary keratinocytes produced comparable amounts of ColVII ( Figures 3C, 3D, and S3E-S3I), suggesting that low levels of ColVII observed in organoids are model dependent. Remarkably, skin organoids did not show obvious morphological defects in the EDJ area, which would be expected in light of the weak anchoring of the epidermis. This could be explained by the lack of exposure to mechanical stress; furthermore, low mechanical stress could by itself affect the assembly of collagen fibrils (Kubow et al., 2015). It would be interesting to determine whether aberrant expression of ColVII could be resolved by maturation and/or changes in mechanical stress; analysis of grafted organoids could be informative in this regard. Importantly, the aberrant levels of ColVII could be a limiting factor for the use of skin organoids for in vitro disease modeling: its low expression does not only result in severe types of EB, but ColVII also plays an important role in wound healing and epidermal squamous cell carcinoma (Chung and Uitto, 2010;Nyström et al., 2013).
Basal keratinocytes in skin organoids adhere to the BM via type I HDs and integrin b1-based adhesion complexes Firm adhesion of keratinocytes to the BM is mediated by HDs, which link the keratin cytoskeleton to the laminin-332 via the integrin a6b4. Since impaired function of virtually any HD component leads to the development of EB, we thought it was imperative to investigate to what extent skin organoids form HDs (Margadant et al., 2010). Using TEM, we detected numerous HDs localized at the basal membrane of the keratinocytes in EDJ ( Figure 4A). Next, we investigated the expression of BP230, a plakin that, in addition to plectin, connects integrin a6b4 to keratin cytoskeleton in more complex type I HDs, which are normally found in the epidermis (Margadant et al., 2010). We observed colocalization of BP230 with HD components a6b4, plectin, and laminin-332, confirming the presence of type I HDs in organoids ( Figure 4B).
Integrins consisting of b1 subunits, particularly a3b1 and a2b1, also contribute to the adhesion of basal keratinocytes by linking the ECM to the actin cytoskeleton via adaptor proteins talin and vinculin (Margadant et al., 2010). Whereas vinculin is restricted to the BM in adult skin, its expression can also be observed at the lateral and apical membranes of basal and suprabasal keratinocytes in skin organoids ( Figure 4C); this resembles vinculin localization in skin at mid-gestation of human fetal development ( Figure S4) (Hentula et al., 2001). Integrin a3b1and a2b1-containing adhesion structures also play a role in the migration and spreading of keratinocytes during nonhomeostatic conditions, such as wound healing, as well  (legend continued on next page) as in maintaining cell-cell contact integrity (Zuidema et al., 2020). Keratinocytes in epidermis form cell-cell adhesions via E-cadherin (E-cad) + adherens junctions, desmoglein-3 (dsg-3) + desmosomes, zonula occludens-1 (ZO-1) + tight junctions, and connexin 43 (Cx43) + gap junctions (Churko and Laird, 2013;Sumigray and Lechler, 2015). All main adhesion junctions were observed in skin organoids at expected sites (Figures 4A and 4D-4F), further confirming the relatively mature state of skin stratification.
In summary, we were able to generate hair-bearing skin organoids from two independent hiPSC lines following the multistep protocol recently developed by . Moreover, we thoroughly characterized their EDJ. We showed that skin organoids form a fully stratified interfollicular epidermis in vitro. Basal keratinocytes in organoids adhere to laminin-332 and ColIV-rich BM via type I HDs and integrin b1-based adhesion complexes. The EDJ in organoids derived from our cell lines was almost devoid of ColVII, indicating that further maturation is required to take full advantage of skin organoids as disease model for some forms of EBs, in particular those caused by mutations in the COL7A1 gene (Table S1).

Generation of skin organoids and quantification of HF-formation frequencies
Hair-bearing skin organoids were generated as previously described (Lee and Koehler, 2020). Skin organoids that did not show bipolar organization at around day 30 were excluded from the study. Quantifications of the percentage of organoids producing HFs and of the number of HFs per skin organoid were performed at day 130 in 3 independent differentiations for both hiPSC lines, respectively. Images of each organoid were taken with a Leica M420 macroscope using an Olympus XC50 digital color camera. The angle of the organoids revealing the highest number of HFs was selected for quantification.

Human tissue specimens
Adult human skin samples used in this study were obtained upon mammoplasty or abdominoplasty of females of 32-to 40-years-old with type I/II skin, based on the Fitzpatrick skin-type classification scale. The human fetal tissue used in this work (19 weeks, facial area) was obtained from elective abortion (without medical indication) with signed informed consent from all donors. The work described here was reviewed and approved by the Medical Ethical Committee of Leiden University Medical Center (P08.087).

Antibodies
Primary antibodies used are listed in Table S2, and secondary antibodies are listed in supplemental experimental procedures.

IF microscopy
For cryosections, organoids were embedded in 15% sucrose/7.5% gelatin solution as previously described (Bajanca et al., 2004). Sections (10 mm thick) were fixed for 10 min in ice-cold acetone (or 15 min in 4% paraformaldehyde when stained with Nile Red) and blocked with 2% bovine serum albumin (BSA; Sigma) in PBS for 1 h at room temperature (RT). hiPSC-derived and primary keratinocytes were seeded on coverslips and fixed after 4 days with 2% paraformaldehyde for 30 min, permeabilized with 0.2% TritonX-100 for 5 min, and blocked with PBS 2% BSA for 1 h at RT. Incubations with primary and secondary antibodies were performed in PBS 2% BSA for 1 h at RT. For Nile Red staining, sections were further incubated for 15 min in Nile Red/PBS (0.05 mg/mL). Nuclei were stained with DAPI. When indicated, filamentous actin was visualized using Alexa Fluor 647-conjugated phalloidin (Thermo Fisher Scientific, #A22287). Sections were mounted in Fluoromount-Gâ (SouthernBiotech) and coverslips in Mowiol. For whole-mount immunostaining, organoids were processed as (C) Representative confocal images of organoid and human skin sections stained with anti-a6 integrin, anti-laminin-332, and antivinculin antibodies. Whereas vinculin is restricted to the BM in the adult skin, its expression can be observed also at the lateral and apical membranes of basal and suprabasal keratinocytes in skin organoids (arrowhead), which is similar to that observed during the midgestation period of human fetal development ( Figure S4). (D-F) Representative confocal images of skin organoids stained for desmosomal marker-desmoglein-3 (dsg-3) and integrin a6 together with (D) gap junction marker connexin 43 (Cx43), (E) adherens junction marker E-cadherin (E-cad), or (F) tight junction marker ZO-1. Note the lower expression of dsg-3 in the upper layers of epidermis. Dsg-3, Cx43, E-cad, and ZO-1 localize to the cell-cell junctions. Dashed boxes are magnified to the right. (B-E) Scale bars: 40 mm. Confocal images are presented as maximum projected z stacks. All organoids are imaged at day 130.
previously described . Cryosections (human skin and day-130 organoids) and keratinocytes were analyzed with a Zeiss LSM900 Airyscan2 upright confocal microscope, and cryosections of day-55 organoids as well as whole mounts were analyzed with a Leica TCS SP5 confocal microscope. Images of maximum intensity projection are presented. Images of skin organoids developed from the LUMCi045-A1 hiPSC line are shown. All images were processed using Fiji/ImageJ (Rueden et al., 2017;Schindelin et al., 2012). Detailed explanation of the quantification of the ECM components can be found in supplemental experimental procedures.

Histology
Cryosections of skin organoids were stained for hematoxylin and eosin (H&E). Images were taken with an Olympus AX70 microscope using an Olympus XC50 digital color camera.

TEM
For TEM, tissue samples were fixed in Karnovsky's fixative. Post-fixation was done with 1% osmiumtetroxide in 0.1 M cacodylatebuffer. Organoids were washed and stained en bloc with Ultrastain 1 (Leica, Vienna, Austria), followed by ethanol dehydration series. The samples were embedded in a mixture of DDSA/NMA/Embed-812 (EMS, Hatfield, PA, USA), sectioned, and analyzed with a Tecnai12G2 electron microscope (Thermo Fischer Scientific, Eindhoven, the Netherlands). Images of skin organoids developed from the LUMCi045-A1 hiPSC line are shown.

Statistical analysis
Statistical analysis was performed using GraphPad Prism (v.9.1.1). Unpaired two-tailed t test was used to compare two experimental groups. Experiments with more than two experimental groups were analyzed using one-way ANOVA. Comparisons were conducted using Tukey's multiple comparison test after a global ANOVA was determined to be significant. Results with p values lower than 0.05 were considered significantly different from the null hypothesis.

AUTHOR CONTRIBUTIONS
K.R. and V.R. designed and performed the experiments and wrote the manuscript. H.J. performed TEM, I.F. provided samples for the generation of hiPSC lines, A.P. and W.R. provided primary keratinocytes, and S.M.C.d.S.L. collected and isolated the fetal human skin. K.R., C.F., X.G., and C.L.M. provided supervision and discussion. All authors read and approved the manuscript.

CONFLICTS OF INTEREST
C.L.M. is associate editor of Stem Cell Reports.

ACKNOWLEDGMENTS
We would like to thank T. Hashimoto (Osaka City University Graduate School of Medicine) and H. Herrmann (University Hospital Er-langen) for sharing antibodies, A. Sonnenberg (The Netherlands Cancer Institute) for sharing reagents and for critical reading of the manuscripts, Lennard Voortman (Leiden University Medical Center) for technical support with confocal microscopy, and colleagues in the Department of Dermatology, Leiden University Medical Center, for providing adult skin samples. We would like to thank the staff of the Center for Contraception, Abortion and Sexuality (Leiden and The Hague) for collection and provision of fetal material used in this work as well as the donors. This work was supported by DEBRA Austria and by the Novo Nordisk Foundation, Denmark (grant number NNF21CC0073729). K.R. is Chargé de Recherche at the Institut National de la Santé et de la Recherche Médicale (INSERM).